Common and divergent features in transcriptional control of the homologous small RNAs GlmY and GlmZ in Enterobacteriaceae

Abstract

Small RNAs GlmY and GlmZ compose a cascade that feedback-regulates synthesis of enzyme GlmS in Enterobacteriaceae. Here, we analyzed the transcriptional regulation of glmY/glmZ from Yersinia pseudotuberculosis, Salmonella typhimurium and Escherichia coli, as representatives for other enterobacterial species, which exhibit similar promoter architectures. The GlmY and GlmZ sRNAs of Y. pseudotuberculosis are transcribed from σ(54)-promoters that require activation by the response regulator GlrR through binding to three conserved sites located upstream of the promoters. This also applies to glmY/glmZ of S. typhimurium and glmY of E. coli, but as a difference additional σ(70)-promoters overlap the σ(54)-promoters and initiate transcription at the same site. In contrast, E. coli glmZ is transcribed from a single σ(70)-promoter. Thus, transcription of glmY and glmZ is controlled by σ(54) and the two-component system GlrR/GlrK (QseF/QseE) in Y. pseudotuberculosis and presumably in many other Enterobacteria. However, in a subset of species such as E. coli this relationship is partially lost in favor of σ(70)-dependent transcription. In addition, we show that activity of the σ(54)-promoter of E. coli glmY requires binding of the integration host factor to sites upstream of the promoter. Finally, evidence is provided that phosphorylation of GlrR increases its activity and thereby sRNA expression.

Figure 1.

Organization of the glmY and glmZ genes in Enterobacteriaceae. (A) Diagram illustrating gene synteny of the glmY and glmZ regions in Enterobacteriaceae. The gene cluster glmY-glrK-yfhG-glrR-glnB is conserved in Enterobacteriacea, but in some species e.g. Yersinia and Photorhabdus, gene nadE is inserted between glrR and glnB. Upstream of glmY, genes mltF and purL are present except for Providencia sp. Small orfs of unknown function are interspersed between purL and glmY in Yersinia, Photorhabdus and other species. Gene glmZ clusters with the downstream located and divergently orientated hemCDXY cluster, while the region upstream is variable. (B) Organization of enterobacterial glmY and glmZ promoters. Sequence alignments of the glmY and glmZ promoter regions from 39 enterobacterial genomes classified the species into three groups, for which Y. pseudotuberculosis, S. typhimurium and E. coli are representatively shown (for details, see Supplementary Figures S3 and S4). Yersinia possesses the sequences for a σ⁵⁴-promoter (labeled in red) and three GlrR binding sites upstream of both sRNA genes, while overlapping σ⁷⁰-promoters appear to be absent. GlrR binding sites and σ⁵⁴-promoters are also detectable upstream of both sRNA genes in Salmonella, but in addition putative σ⁷⁰-promoters (labeled in blue) that overlap the σ⁵⁴-promoters, are detectable. This arrangement is also found upstream of E. coli glmY. However, E. coli glmZ appears to be transcribed from a single σ⁷⁰-promoter. The sequence alignment also detected two putative IHF binding sites that coincide with the occurrence of σ⁵⁴-promoters.

Figure 2.

Comparison of the roles of GlrR and σ⁵⁴ for expression of glmY from E. coli, S. typhimurium and Y. pseudotuberculosis. (A) EMSAs to test binding of E. coli GlrR protein to the glmY promoter regions of E. coli (−238 to +22), S. typhimurium (−242 to +22) and Y. pseudotuberculosis (−257 to +22). In addition to the glmY promoter fragments, 400 bp (panels 1 and 2) or 200 bp DNA fragments (panel 3) covering the lacZ promoter were present as internal controls. The sizes of the DNA size standard are given at the left. The apparent K_D values are 360 nM for the E. coli glmY promoter, 230 nM for the Salmonella glmY promoter and 290 nM for the Y. pseudotuberculosis glmY promoter. (B) β-Galactosidase activities of E. coli strains carrying fusions of glmY’ from E. coli, S. typhimurium and Y. pseudotuberculosis to the lacZ reporter gene. In addition, these strains had the genotypes indicated in the legend. The following strains and transformants were tested (corresponding to the columns from left to right): Z197, Z206, Z206 + pBGG223, Z206 + pYG6, Z227, Z388, Z389, Z389 + pBGG223, Z389 + pYG6, Z446, Z362, Z363, Z363 + pBGG223, Z363 + pYG6 and Z444.

Figure 3.

Comparison of the roles of GlrR and σ⁵⁴ for expression of glmZ from E. coli, S. typhimurium and Y. pseudotuberculosis. (A) EMSAs to test binding of E. coli GlrR protein to the glmZ promoter regions of E. coli (−424 to +32), S. typhimurium (−242 to + 22) and Y. pseudotuberculosis (−303 to +22). The apparent K_D values for binding of GlrR to the S. typhimurium and Y. pseudotuberculosis glmY promoter fragments are 370 nM in both cases. (B) β-Galactosidase activities of E. coli strains carrying fusions of glmZ’ from E. coli, S. typhimurium and Y. pseudotuberculosis to the lacZ reporter gene. In addition, these strains had the genotypes indicated in the legend. The following strains and transformants were tested (corresponding to the columns from left to right): Z360, Z361, Z361 + pBGG223, Z361 + pYG6, Z443, Z390, Z391, Z391 + pBGG223, Z391 + pYG6, Z447, Z364, Z365, Z365 + pBGG223, Z365 + pYG6 and Z445.

Figure 4.

Transcription of Y. pseudotuberculosis glmZ depends on binding of GlrR to its three target sites upstream of the promoter. (A) β-Galactosidase activities of E. coli strains carrying mutated GlrR binding sites in the chromosomal Y. pseudotuberculosis glmZ’-lacZ fusion. In order to monitor activation by the cognate GlrR protein, Y. pseudotuberculosis glrR was expressed from plasmid pYG6, while the endogenous glrR gene was deleted. The nucleotide exchanges introduced into the ABS are depicted at the left. The following strains were employed (corresponding to the columns from left to right): Z365, Z397, Z398, Z399 and Z400. (B) EMSAs to monitor binding of E. coli GlrR to DNA fragments covering the Y. pseudotuberculosis glmZ promoter and carrying mutations in the ABS as depicted in the Figure.

Figure 5.

Analysis of the E. coli glmZ promoter (A) Schematic representation of the aslA-hemY intergenic region comprising the E. coli glmZ gene. DNA fragments extending until position +32 relative to the glmZ start site and with the 5′ ends indicated by arrows were fused to lacZ. The sequence of the glmZ promoter region with the putative −35/−10 motifs of a σ⁷⁰-promoter is shown below. The nucleotide exchanges that were introduced into these motifs and tested in (C) are marked with asterisks. (B) 5′→3′ deletion analysis of the E. coli glmZ upstream region. β-Galactosidase activities of E. coli wild-type strain R1279 carrying the gradually 5′ truncated glmZ’-lacZ fusions on plasmids. The following plasmids were tested (corresponding to the columns from left to right): pKEM04, pBGG59, pBGG111, pBGG112, pBGG113, pBGG114, pBGG170 and pBGG135. (C) Mutational analysis of the glmZ promoter. The putative −35 and −10 sequences were mutated as indicated in (A) in the context of the glmZ’ (−40 to +32)-lacZ fusion. Plasmids pBGG114, pBGG157 and pBGG171 (corresponding to the columns from left to right) were introduced into wild-type strain R1279 and the β-galactosidase activities were determined.

Figure 6.

Role of IHF for expression of glmY. (A) Schematic representation of the E. coli glmY promoter region and location of GlrR and putative IHF binding sites. The sequences of the putative IHF binding sites upstream of E. coli glmY and Y. pseudotuberculosis glmZ are shown and the nucleotide exchanges introduced in IHF site 1 of the E. coli glmY promoter are indicated. (B) EMSAs to test binding of purified IHF to the glmY and glmZ promoter regions of E. coli and Y. pseudotuberculosis, respectively. The DNA fragments were obtained by PCR making use of the primer pairs BG377/BG456 and BG700/BG701, respectively. As controls, DNA fragments encompassing the lac promoter were additionally present. (C) Expression of E. coli glmY in ΔihfA and ΔihfB mutants. β-Galactosidase activities of strains carrying the chromosomal E. coli glmY’-lacZ fusion in the context of the wild-type promoter (columns 1–3) or in the context of the mutated σ⁷⁰-promoter leaving the σ⁵⁴-promoter as single active promoter (columns 4–6). Genes ihfA or ihfB were deleted as indicated in the legend. The following strains were tested (corresponding to the columns from left to right): Z197, Z395, Z393, Z190, Z394 and Z392. (D) Mutational analysis of the putative IHF site 1 in the E. coli glmY promoter region. β-Galactosidase activities of wild-type and ΔglrR E. coli strains carrying the wild-type or mutated alleles of the E. coli glmY’-lacZ fusion. Mutations were either in the putative IHF-site 1 (columns 3, 4, 7, 8) as indicated in (A) or in the −10 sequence of the glmY promoter (columns 5–8) rendering glmY’-lacZ expression fully dependent on σ⁵⁴. The following strains were employed (corresponding to the columns from left to right): Z197, Z206, Z370, Z372, Z190, Z196, Z371 and Z373.

Figure 7.

Phosphorylation increases activity of response regulator GlrR. (A) Effect of acetyl phosphate on the DNA binding activity of GlrR as revealed by EMSA. EMSAs were performed using purified E. coli GlrR and the E. coli glmY promoter fragment. To test the possible effect of phosphorylation on GlrR activity, the protein was pre-incubated at 37°C for 1 h in the absence (left panel) or presence (right panel) of 50 mM acetyl phosphate before continuing with the EMSA protocol. (B) A glutamate replacement of the phosphorylation site Asp56 in GlrR strongly up-regulates glmY expression. E. coli strain Z206 carrying a ΔglrR mutation and the E. coli glmY’-lacZ fusion on the chromosome was complemented with plasmids carrying E. coli wild-type glrR (pBGG389, column 2), glrR-D56A (pBGG398, column 3), glrR-D56E (pBGG399, column 4) or no gene (pBAD33, column 1) under P_Ara promoter control. Subsequently, the β-galactosidase activities were determined from these transformants.

Figure 8.

Model illustrating the roles of the TCS GlrR/GlrK, σ⁵⁴ and IHF for transcription of sRNA genes glmY and glmZ in Enterobacteriaceae. Histidine kinase GlrK phosphorylates response regulator GlrR, which stimulates binding of GlrR to its target sites on the DNA. GlrR binds to three activator binding sites present upstream of σ⁵⁴-dependent promoters that control the expression of sRNA genes glmY in all species and glmZ in a subset of species. GlrR, which contains a σ⁵⁴ interaction domain, is absolutely required for activity of these σ⁵⁴-promoters. In addition, promoter activity depends on IHF, which might facilitate interaction of GlrR with the σ⁵⁴-RNA polymerase by binding-induced bending of the promoter DNA. In Y. pseudotuberculosis, transcription of glmY and glmZ is directed by single σ⁵⁴-promoters that require activation by GlrR. Hence, glmY and glmZ compose a regulon controlled by GlrR and σ⁵⁴. A similar arrangement is found in S. typhimurium, but σ⁷⁰-promoters that overlap the σ⁵⁴-promoters additionally contribute to glmY and glmZ expression. Overlapping σ⁵⁴- and σ⁷⁰-promoters also direct expression of the E. coli glmY gene, while expression of glmZ is achieved from a single constitutively active σ⁷⁰-promoter. Sequence alignment analyses suggest that these three different arrangements might also apply to other enterobacterial species as shown in the Figure.